Until about a decade ago, resolution in far-field light microscopy was thought to be limited to 200-250 nanometers in the focal plane, concealing details of sub-cellular structures and constraining its biological applications. Breaking this diffraction barrier by the seminal concept of stimulated emission depletion (“STED”) microscopy has made it possible to image biological systems at the nanoscale with light. STED microscopy and other members of reversible saturable optical fluorescence transitions (“RESOLFT”) family achieve a resolution >10-fold beyond the diffraction barrier by engineering the microscope's point-spread function (“PSF”) through optically saturable transitions of the (fluorescent) probe molecules.
However, slow progress in 3D super-resolution imaging has limited the application of prior art techniques to two-dimensional (“2D”) imaging. The best 3D resolution until recently had been 100 nanometers axially at conventional lateral resolution. 4 Pi microscopy achieved this through combination of two objective lens of high numerical aperture, in an interferometric system. 4 Pi microscopy was only recently shown to be suitable for biological imaging. Only lately the first 3D STED microscopy images have been published exceeding this resolution moderately with 139 nanometer lateral and 170 nanometer axial resolutions. While this represents a 5-fold smaller resolvable volume than provided by conventional microscopy, it is still at least 10-fold larger than a large number of sub-cellular components, such as synaptic vesicles, for example. A more recent development achieves 3D resolution below 50 nm in all 3 directions by combining STED with 4 Pi microscopy.
To measure dynamic properties of a biological system, particle-tracking techniques have been developed over the last decades. Particle-tracking techniques can localize small objects (typically less than the diffraction limit) in live cells with sub-diffraction accuracy and track their movement over time by taking a time series of recordings. Single particles are imaged conventionally, with or without total internal reflection illumination, or in a multi-plane arrangement. Every particle produces a diffraction limited image. By determining the center of the blurry image (the width of the intensity distribution is equivalent to the ‘spatial resolution’ of the microscope), the position of the particle can be determined. The spatial localization accuracy of single particles in a fluorescence microscope is the square root of the total number of detected fluorescence photons from the particle in the absence of background and effects due to finite pixel size.
Recently, this concept has also entered the emerging field of super-resolution microscopy. In techniques such as ‘FPALM’, ‘PALM’, ‘STORM’, or ‘PALMIRA’, biological samples are labeled by photoactivatable fluorescent molecules. Only a sparse distribution of single fluorophores is activated, and hence imaged, at any time by a sensitive camera. This allows spatial separation of the diffraction-limited intensity distributions of practically every fluorescing molecule and localization of individual fluorophores with accuracy typically in the 10 nm range (standard deviation σ). By bleaching or deactivating the fluorescing molecules during the read-out process and simultaneously activating additional fluorophores, a large fraction of the probe molecules are imaged over a series of many image frames. A super-resolved image at typically 20-30 nm resolution (measured as the FWHM of a distribution; ˜2.4σ) is finally assembled from the determined single molecule positions.
Recently, particle-tracking of sub-cellular fluorescent components and localization-based super-resolution microscopy techniques have advanced from a two-dimensional (2D) imaging method to the third dimension. Localization in the z-direction is complicated by the fact that camera images are 2D. Different z-positions do not result in easily detectable shifts of the center of mass as it is in the 2D case. The axial position has to be deduced from the defocused 2D intensity distributions taking the complex dependence of the focal intensity distribution in the axial direction into account. Analyzing the diameter of the rings appearing in the defocused images, for example, allows conclusions on its z-position. A major obstacle is the axial symmetry of the intensity distribution (in a perfect microscope): for an observed 2D image an axial position of z0 is equally possible as −z0. To break this symmetry, multi-plane detection has been developed.
Recording images in different focal planes simultaneously provides means to determine the axial position of a particle uniquely. This multi-plane detection approach has successfully been used in slightly varying arrangements to track particles down to single quantum dots within cells and has been recently applied to localization-based 3D super-resolution microscopy.
The context of morphology and movement of a biological particle or structure with regard to other structures in a cell can be of high importance. To measure this, typically multiple labels marking different structures (for example two different proteins) by different photo-physical properties (usually two different fluorescence colors) are imaged. Multi-color recordings are used in super-resolution microscopy as well as in particle tracking.
In super-resolution microscopy and particle tracking, small structures featuring only a small number of labels, often only single fluorescent molecules, are observed. Background suppression is therefore of high importance. An often applied method in 2D particle tracking and 2D super-resolution microscopy is illumination at an angle at which the light experiences total internal reflection at the coverslip-specimen interface. The light in this ‘total internal reflection microscopy’ (TIRF) mode can in this case only penetrate on the order of 70 to 200 nm into the specimen (depending on an adjustable incidence angle) and no background light can be created in planes beyond this depth range therefore reducing the amount of light penetration into the sample dramatically.